In the United States, the incidence of gram-negative bacteremia varies between 71,000 and 330,000 cases per year, but despite the advances in antimicrobial therapy, the mortality associated with gramnegative bacteremia (including postsurgical sepsis) remains between 20% and 50% (1). There is evidence that translocating intestinal bacteria cause a significant proportion of complicating infections (including sepsis) in hospitalized immunosuppressed patients, postsurgical patients, and trauma patients (2). Normal intestinal bacteria can initiate systemic disease by translocating through a histologically intact intestinal mucosa, but the routes and mechanisms of bacterial transport have remained elusive due to the complex dynamics of the intestinal tract. Recent in vivo studies have indicated that bacteria can translocate to the draining mesenteric lymph node within tissue macrophages (17, 20-21,117), and translocating Enterococcus faecalis (9) and Candida albicans (17) have been localized within intact intestinal epithelial cells. The primary aim of this proposal is to clarify the initial events in bacterial translocation through an intact intestinal mucosa. Results from in vivo mouse model are correlated with results from in vitro experiments involving the interactions of translocating bacteria with cultured intestinal epithelial cells and with mononuclear phagocytes. Nine different bacterial strains that translocate with varying frequencies (frankly pathogenic strains that translocate with high frequency [Salmonella typhimurium, Listeria monocytogenes], facultative strains that translocate with intermediate frequency [Escherichia coli, Proteus mirabilis, Enterococcus faecalis], anaerobic strains that rarely translocate [Bacteroides fragilis]) are studied in conventionally reared and germfree mice; translocation is monitored by quantitative microbiological methods, as well as light, immunofluorescent, and electron microscopic methods for visualization of the route of translocation within the intestinal mucosa. These in vivo results are correlated with the results of in vitro experiments designed to dissect the factors involved in the interactions (adherence, uptake, intracellular survival) of these bacterial strains with cultured enterocytes and with mononuclear phagocytes. The results of in vitro and in vivo experiments can be correlated to clarify the roles of the enterocyte and the tissue macrophage in the process of bacterial translocation. This approach will not only clarity the routes and mechanisms involved in bacterial translocation, but should also (a) contribute to our understanding of the normal function of intestinal cells; (b) elucidate the role of the enterocyte as a nonprofessional phagocyte; (c) clarify the interactions of normal enteric bacteria with tissue macrophages; (d) expand our knowledge of bacterial virulence mechanisms at the cellular level; (e) suggest, most likely, new treatment regimens to decrease the costly morbidity associated with systemic infections caused by translocating bacteria.